Sufficient engagement of CD4+ T cells (TCD4+) is critical for a positive outcome in several human infections, including influenza (flu). TCD4+ are activated by complexes of pathogen-derived peptides (epitopes) and major histocompatibility class II molecules (MHCII) that are generated within the antigen bearing cell and then transported to the cell surface where they can be engaged by T cell receptor. According to convention, peptide-MHCII complexes are formed following internalization of exogenous antigen, proteolysis within the endocytic compartment and loading onto nascent MHCII in a late endosomal compartment. This classical pathway has been deduced mainly through study of durable globular proteins. When infectious agents are used, additional pathways of peptide generation (antigen processing) become apparent, some that begin with endogenous sources of antigen (synthesized within the presenting cell). A critical question that we have begun to address is the extent to which these alternative pathways drive the total TCD4+ response to a bona fide pathogen. Using a C57Bl/6 (B6) model of influenza infection, preliminary results indicate that an astonishing 13 of the 14 epitopes that account for the TCD4+ response can be generated non-classically. Thus, this line of investigation could dramatically revise a fundamental principle of basic immunology. Organized in four independent but highly integrated specific aims, the work proposed here will solidify and expand upon these findings, investigate the relative capacities of the different professional APCs (dendritic cells, macrophages and B cells) for alternative processing of flu proteins during primary and secondary responses, and determine whether the processing pathway utilized to generate an epitope can impact the level of TCD4+ functionality and, hence, protective capacity. The results of these studies could considerably impact the rational design of vaccines against an array of pathogens. In addition, they could point to new approaches to cancer immunotherapy, and provide important insight into the genesis and treatment of autoimmune diseases.